Chemical Senses, 2015, Vol 00, 1–13doi:10.1093/chemse/bjv049
Original Article
Original Article
Cre-Mediated Recombination in Tas2r131 Cells—A Unique Way to Explore Bitter Taste Receptor Function Inside and Outside of the Taste SystemAnja Voigt1,2, Sandra Hübner1, Linda Döring1, Nathalie Perlach1, Irm Hermans-Borgmeyer3, Ulrich Boehm2,4 and Wolfgang Meyerhof1
1Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114–116, 14558 Nuthetal, Germany, 2Institute for Neural Signal Transduction, Center for Molecular Neurobiology Hamburg, Falkenried 94, 20251 Hamburg, Germany and 3Transgenic Animals Service Group, Center for Molecular Neurobiology Hamburg, UKE, Martinistraße 52, 20246 Hamburg, Germany 4Present address: Department of Pharmacology and Toxicology, University of Saarland, School of Medicine, 66421 Homburg, Germany
Correspondence to be sent to: Wolfgang Meyerhof, Department of Molecular Genetics, German Institute of Human Nutri-tion Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114–116, 14558 Nuthetal, Germany. e-mail: [email protected] Boehm, Department of Pharmacology and Toxicology, University of Saarland, Kirrberger Strasse Building 64.1, 66421 Homburg, Germany. e-mail: [email protected]
Accepted 18 August 2015.
Abstract
The type 2 taste receptors (Tas2rs) comprise a large family of G protein-coupled receptors that recognize compounds bitter to humans and aversive to vertebrates. Tas2rs are expressed in both gustatory and nongustatory tissues, however, identification and functional analyses of T2R-expressing cells have been difficult in most tissues. To overcome these limitations and to be able to manipulate Tas2r-expressing cells in vivo, we used gene-targeting to generate a Tas2r131-specific Cre knock-in mouse strain. We then employed a binary genetic approach to characterize Cre-mediated recombination in these animals and to investigate Tas2r131 expression during postnatal development. We demonstrate that a Cre-activated fluorescent reporter reliably visualizes Tas2r131-cells in gustatory tissue. We show that the onset of Tas2r131 as well as of α-Gustducin expression is initiated at different developmental stages depending on the type of taste bud. Furthermore, the number of Tas2r131- and α-Gustducin-expressing cells increased during postnatal development. Our results demonstrate that the Tas2r131-expressing cells constitute a subpopulation of α-Gustducin positive cells at all stages. We detected Tas2r131-expressing cells in several nongustatory tissues including lung, trachea, ovary, ganglia, and brain. Thus, the Tas2r131-Cre strain will help to dissect the functional role of Tas2r131 cells in both gustatory and nongustatory tissues in the future.
The type 2 taste receptors (Tas2rs; a.k.a. bitter taste receptors) com-prise a large family of G protein-coupled receptors (GPCRs) encoded
by the Tas2r genes. Functional Tas2r gene numbers vary depend-ing on the species with 25 genes in humans and 35 genes in mice (Go et al. 2005). Tas2rs are glycoproteins that function as receptors
Chemical Senses Advance Access published September 16, 2015 by guest on N
for the structurally diverse, natural, and synthetic bitter-tasting sub-stances (Adler et al. 2000; Chandrashekar et al. 2000; Matsunami et al. 2000; Reichling et al. 2008; Meyerhof et al. 2010; Thalmann et al. 2013). Recognition of the countless bitter substances is mostly achieved through Tas2rs with broad agonist spectra (Meyerhof et al. 2010; Behrens et al. 2014). Tas2rs were first described in the gus-tatory system, where subsets of them are coexpressed in the same cells (Adler et al. 2000; Matsunami et al. 2000; Behrens et al. 2007). These cells also contain the G protein α-gustducin (α-Gust) and constitute a subpopulation of taste receptor cells (TRCs; a.k.a. type II cells) (Adler et al. 2000; Chandrashekar et al. 2000; Matsunami et al. 2000; Kim et al. 2003).
Tas2r expression is not restricted to the gustatory system. Several studies have reported Tas2r expression in extra-oral tissues, includ-ing the gastrointestinal (GI) tract and the respiratory system (Wu et al. 2002; Finger et al. 2003; Tizzano et al. 2010; Behrens and Meyerhof 2011; Tizzano et al. 2011; Prandi et al. 2013; Krasteva-Christ et al. 2015). Hence, Tas2r-meditated responses do not seem to be limited to tissues with direct access to the environmental com-pounds such as the GI tract or respiratory tissues, in which Tas2rs have been proposed to respond to ingested and inhaled toxins (Tizzano et al. 2010; Jeon et al. 2011). Tas2rs are also expressed in the nervous system, heart, testes, immune system, and thyroid gland, that is, tissues which are not directly accessible for environmental substances (Singh et al. 2011; Dehkordi et al. 2012; Li and Zhou 2012; Voigt et al. 2012; Foster et al. 2013; Clark et al. 2015; Voigt et al. 2015). Proposed functions in these tissues include modulation of neurotransmission, neuronal food intake regulation, monitoring of cardiac nutrient sensing, and contributing to the inhibition of TSH-mediated iodine secretion (Singh et al. 2011; Foster et al. 2013; Clark et al. 2015; Voigt et al. 2015).
Although Tas2rs are involved in more than taste, functional anal-ysis of Tas2rs in extra-oral tissues has however remained challeng-ing. This is at least partially due to the fact, that Tas2rs—as many other GPRCs—are expressed at low levels (Fredriksson and Schiöth 2005; Regard et al. 2008). Furthermore, only one Tas2r-specific antiserum (against TAS2R38) has been reported this far to spe-cifically label the respective human Tas2r cell population (Behrens et al. 2012). The same antiserum was also used to visualize murine Tas2r138—which is the murine orthologue of the human TAS2R38 gene—cells (Jeon et al. 2008), however, later thorough characteriza-tion of this antibody revealed that it is exclusively specific to humans (Behrens et al. 2012). Taken together, both low Tas2r expression lev-els and the absence of reliable antisera against most Tas2rs hamper
the identification of Tas2r-expressing cells and thus functional analy-sis in extra-oral tissues. To overcome these limitations, we recently developed a genetic approach to visualize Tas2r131-expressing cells in mice (Voigt et al. 2012). In this mouse strain, the open reading frame of the Tas2r131 gene was replaced with an expression cas-sette containing a fluorescent reporter protein. These mice enabled us to visualize and quantify the entire oral Tas2r131 cell popula-tion (Voigt et al. 2012). Tas2r131 cells were also detected in a few extra-oral tissues, including the vomeronasal organ, nasal epithelia, thymus, testis, epididymis, and sperm (Voigt et al. 2012).
To be able to actually manipulate Tas2r131-expressing cells in vivo, we used, in the present report, gene-targeting to generate a Tas2r131-specific Cre knock-in mouse strain and employed a binary genetic approach to characterize Cre-mediated recombination in these animals and to investigate Tas2r131 expression in gustatory and nongustatory tissues during postnatal development.
Materials and methods
Construction of the Tas2r131-barley lectin-IRES-Cre (Tas2r131-BLiC) targeting vectorA BL-IRES-Cre (BLiC) expression cassette was inserted into the previously described Tas2r131 targeting vector (Voigt et al. 2012) containing 5′ (1.8 kb) and 3′ (1.9 kb) fragments of the Tas2r131 gene (MGI:2681280). Sequences encoding both flag and c-myc epitopes (for differential transsynaptic tracing) were inserted into the BL sequence at position 79 and 597. The unique BglII site 3′ of the poly(A) signal of the Cre cDNA was used to insert an AFN (tACE-Flpe/NEOr) neomycin selection cassette, flanked by FRT sites. Sequencing confirmed the accuracy of the final targeting vector.
Gene-targetingThe Tas2r131-BLiC targeting vector was linearized with AscI and electroporated into R1 ES cells (Nagy et al. 1993). G418-resistant colonies were picked and genotyped by Southern blot analysis as described (Voigt et al. 2012). Recombinant ES cell clones were identified using external flanking probes for Southern blot analysis, which were generated by PCR using mouse genomic DNA as tem-plate (Table 1; Tas2r131_upstream_for, Tas2r131_upstream_rev). Correctly targeted ES cells were injected into C57BL/6J blastocysts to generate chimeras that were backcrossed to C57BL/6J animals to obtain heterozygous Tas2r131-BLiC (Tas2r131+/BLiC) mice. In order to remove the neomycin selection cassette, heterozygous Tas2r131+/BLiC
Table 1. List of oligonucleotides
Oligonucleotide Sequence (5′ to 3′) TA (°C) Amplicon size (bp)
mice were subsequently crossed to Flpe-deleter mice (Rodríguez et al. 2000) to remove the selection cassette, and removal of the selection cassette in the resulting offspring was confirmed by PCR (Table 1; AFN_for, AFN_rev).
MiceAnimal care and experimental procedures were performed in accord-ance with the guidelines established by the animal welfare committees of the University of Hamburg (Hamburg, Germany; Permit Number: G 21307/591-00.32, No. 11/07) and of the Ministry of Environment, Health and Consumer Protection of the federal state of Brandenburg (State of Brandenburg, Germany, Permit Number: 23-2347-A-1-1-2010). Mice were housed under standard light/dark cycle with water and food ad libitum. Tas2r131+/BLiC mice were interbred to obtain mice homozygous for the recombinant Tas2r131 allele (Tas2r131BLiC/BLiC). To monitor Cre recombinase activity, Tas2r131+/BLiC mice were crossed with a fluorescent reporter mouse strain (eR26-τGFP) (Wen et al. 2011) to generate Tas2r131+/BLiC/eR26+/τGFP double knock-in mice. Tas2r131BLiG/BLiG animals were described before (Voigt et al. 2012) and were backcrossed for 10 generations to C57BL/6 mice. The Tas2r131BLiC line was used previously (Foster et al. 2013; Prandi et al. 2013; Clark et al. 2015; Voigt et al. 2015) and both Tas2r131BLiC and Tas2r131+/BLiC/eR26+/τGFP mice were kept in a mixed (129/SvJ and C57BL/6) background. Mice of either sex were used in the experiments. Littermates carrying Tas2r131 wild type (wt) alleles and C57BL/6 mice were used as controls.
GenotypingGenomic DNA from mouse tail biopsies was obtained by protein-ase K (Roth) digestion and isolated using the Phire Animal Tissue Direct PCR Kit (Finnzymes, Fischer Scientific) as recommended by the manufacturer. Subsequently, PCR reactions were performed to identify either wt (Table 1; Tas2r131_wt_for, Tas2r131_wt_rev) or knock-in alleles in gene-targeted mice (Table 1; Cre_for, Cre_rev). To confirm the presence of the ROSA26 knock-in allele we used primers Rosa26_for and Rosa26_rev (Table 1).
Tissue preparationFor in situ hybridization (ISH) and immunofluorescence (IF) analy-ses, tissues were prepared from transcardially perfused mice at post-natal day (P) 3, at P9, at P17, as well as from adult (10−26 weeks old) animals. Postfixation, cryoprotection, and freezing of tissues were done as described (Voigt et al. 2012). However, P3 animals were kept undissected for both the postfixation and the cryoprotec-tion step. Serial 10 or 14 µm thick sections were generated using a cryostat (Thermo Scientific) and mounted onto SuperFrost Plus glass slides (Thermo Scientific). Sections of P3 animals were obtained by dissecting the entire P3 animal. For RNA analysis, tissues were obtained from nonperfused adult mice and immediately frozen in liquid nitrogen.
Reverse transcription polymerase chain reactionRNA was extracted from mouse tissues using TRIzol reagent (Invitrogen) and DNaseI (Invitrogen) digestion was performed according to manufacturer’s protocol. Subsequently, cDNA synthesis was carried out using Superscript II reverse transcriptase and ran-dom hexamers (Invitrogen). Omitting reverse transcriptase served as negative control. cDNAs corresponding to 10 ng of reverse tran-scribed RNA and TITANIUM TaqDNA-polymerase (Clonetech) were subjected to the PCR reactions (40 cycles). A Tas2r131-specific
amplicon was obtained using oligonucleotides Tas2r131_for and Tas2r131_rev, Cre-specific PCR products were produced using primers Cre_for and Cre_rev (Table 1). The housekeeping β-actin gene was used as control (β−actin_for, β−actin_rev; Table 1).
Generation of probes for ISHThe preparation of digoxigenin-labeled Tas2r131 antisense (as) and sense riboprobes has been described before (Voigt et al. 2012). The Cre recombinase-specific RNA probe was generated by PCR using primers Cre_for and Cre_rev (Table 1), TITANIUM TaqDNA-polymerase (Clontech) and template DNA (the GnRHR-IRES-Cre (Wen et al. 2008) targeting vector). Cre recombinase-specific PCR fragments were subcloned into pBlueScript I KS− (Stratagene) and linearized with EcoRI (Cre as) or NotI (Cre sense) (Fermentas). In vitro transcriptions were performed in the presence of digoxigenin-labeled ribonucleotides (Roche Applied Science) to generate the probes. 50 U of RNase Inhibitor (Fermentas) were added to the transcription reaction. To remove genomic DNA, RNA samples were incubated with DNase I (Invitrogen) for 30 min at 37 °C. Subsequently, probes were purified by ammonium chloride/ethanol precipitation overnight at −20 °C.
In situ hybridizationBoth colorimetric and fluorescent ISH were performed as described (Voigt et al. 2012). Briefly, 10 µm thick sections were postfixed (4% PFA), washed (PBS), and permeabilized (1% Triton-X100). Next, a proteinase K (20 µg/mL) digestion step was performed and the reaction was stopped using glycine (0.2%) and 2 additional wash-ing steps with PBS. Prior to acetylation in 0.1 M triethanolamine buffer, tissue sections were again fixed with 4% PFA. Tissue sec-tions were prehybridized (5 h, 50 °C) in prehybridization solution and then hybridized (overnight, 50 °C) using specific riboprobes. On the following day sections were processed either for colorimetric (Behrens et al. 2007) or fluorescent detection (Voigt et al. 2012). Although hybridization with Cre-specific riboprobes included an RNase treatment (1 µg/mL), no RNase was added to sections treated with the Tas2r131 riboprobes. Sections were finally mounted onto glass coverslips and images were taken using Eclipse E 1000 (Nikon Instruments) or TCS SP2 (Leica; excitation: 488 nm, emis-sion 505−530 nm) microscopes.
ImmunfluorescenceIF was described previously (Voigt et al. 2012). Briefly, 14 µm thick tissue sections were washed (PBS), permeabilized (0.05% Triton-X 100), blocked (TNB), and incubated with the appropriate antiserum (Table 2) for 2 h at room temperature (RT) and then overnight at 4 °C. The anti-NTPDase2 (Bartel et al. 2006), anti-PLC-β2 (Kim et al. 2006), and anti-α-Gust (Caicedo et al. 2003) antisera used in this study have been thoroughly validated in cell lines and taste tissues before. On the next day, slides where washed three times (TNT), treated with secondary antiserum for 2 h at RT, washed again (TNT), and then cover-slipped using a fluorescent mounting medium (Dako). For nuclear staining, sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Sigma) for 10 min before cover-slipping. Negative controls were processed in parallel in every experiment, with primary antisera omitted. No fluorescence was detected. Moreover, we did not observe hrGFP or τGFP fluores-cence in wt mice. Fluorescent images were collected using a TCS SP2 microscope (Leica). To sequentially scan the entire depth of the tis-sue, 0.5 µm scanning steps were performed at excitation wavelengths of 488, 561, and 633 nm, respectively. Emission of the fluorochromes
and the fluorescent proteins hrGFP and τGFP were detected between 505−530, 590−630, and 645−740 nm wavelengths, respectively.
Results
Generation of the Tas2r131-BL-IRES-Cre knock-in miceThe linearized Tas2r131-BLiC targeting construct (Figure 1A) was electroporated into R1 ES cells and 384 neomycin-resistant ES cell clones analyzed by Southern blot using an external flanking probe (Figure 1B). Two correctly targeted ES cell clones were detected cor-responding to a targeting frequency of 0.5%. One targeted ES clone was expanded and injected into C57BL/6J blastocysts to generate Tas2r131+/BLiC mice. The chimeras were backcrossed with C57BL/6J mice and the recombinant Tas2r131 allele was successfully trans-mitted through the germline as confirmed by Southern blot analysis (Figure 1C). Heterozygous mice Tas2r131+/BLiC were then bred with Flpe deleter animals (Rodríguez et al. 2000) to remove the neomy-cin selection cassette. The resulting offspring was backcrossed with C57BL/6J mice to remove the Flpe transgene and subsequently bred inter se to establish the Tas2r131BLiC/BLiC knock-in strain. The result-ing mice were genotyped by PCR (Figure 1D). Here, we present the initial characterization of this novel mouse strain focusing on the
distribution of Tas2r131-expressing during mouse development. Tracing of gustatory pathways via transfer of BL has been described elsewhere (Voigt et al. 2015).
Absence of Tas2r131 taste receptor RNA and presence of Cre recombinase RNA in taste buds of homozygous Tas2r131BLiC/BLiC miceTo characterize expression of the recombinant Tas2r131 allele, we performed reverse transcription polymerase chain reaction (RT-PCR) analyses for Tas2r131 and Cre RNA of taste bud (TB)-enriched tongue epithelium preparations containing fungiform (FuP), foliate (FoP), vallate papillae (VP), the naso-incisior duct (NID), and the soft palate (SP) (Figure 2A). As expected, Cre mRNA was detected in all investigated tissues of Tas2r131BLiC/BLiC but not in wt mice. Moreover, the absence of Tas2r131 RNA indi-cated successful knock-out of the Tas2r131 gene in the animals homozygous for the Tas2r131-BLiC allele (Figure 2A). To visualize the cells expressing Cre RNA, we conducted ISH experiments using cross-sections of VP (Figure 2B). Cre but not Tas2r131 RNA was detected with the appropriate riboprobes in a few cells of VP in Tas2r131BLiC/BLiC animals (Figure 2B). Vice versa, using VP sections of wt mice, a few cells were labeled with the Tas2r131 but not with the Cre riboprobe (Figure 2B). The relative abundance of cells
Figure 1. Generation of the Tas2r131BLiC mouse strain. (A) The targeting vector, the Tas2r131 wt allele, the targeted Tas2r131 allele before (neo+) and after (neo−) removal of the neomycin selection cassette are shown. Restriction sites for BglII as well as the location of the probe for genotyping (probe I) are indicated. The inserted cassette contains the coding sequence for an epitope-tagged version of BL, an IRES element, and the coding sequence for Cre. The AFN (tACE-Flpe/NEOr) selection cassette is flanked by FRT sites. (B) Southern blot: BglII digested ES cell DNA hybridized with probe I identified one heterozygous ES cell clone (wt 6.9 kb, mut 5.7 kb). (C) Southern blot: Genomic DNA of Tas2r131+/BLiC mice after digestion with BglII exhibiting both the recombinant (5.7 kb) and the wt Tas2r131 allele (6.9 kb). (D) PCR products show the genotype of the gene-targeted Tas2r131 animals using specific primers (red arrows in A).
labeled with the Cre riboprobe in the gene-targeted mouse strain was similar to the number of cells labeled by the receptor-specific riboprobe in wt mice. Taken together, our data indicate that tran-scription of the recombinant Tas2r131 allele is similar to that of the Tas2r131 wt allele.
Next we bred the Tas2r131+/BLiC mice with enhanced-Rosa26-tauGFP (eR26-τGFP) reporter animals (Wen et al. 2011), that express a fusion of the microtubule-associated protein τ with GFP (Rodriguez et al. 1999) under control of the CAGS promoter (Lobe et al. 1999) in the ROSA26 locus leading to constitutive and ubiquitous high-level gene expression in Cre-expressing cells. In heterozygous offspring (Tas2r131+/BLiC/eR26+/τGFP), Cre-mediated recombination leads to expression of the reporter τGFP resulting in fluorescently labeled Tas2r131-expressing cells (Figure 3A). Because Cre-mediated recombination is irreversible, Tas2r131+/BLiC/eR26+/τGFP mice report the history of activity of the Tas2r131 promoter. To test whether both the wt and recombinant Tas2r131 alleles are simultaneously expressed in adult mice, we combined ISH with IHC analyses. Figure 3B shows that the same subset of VP taste cells is labeled by the Tas2r131 riboprobe and by an antiserum against τGFP in Tas2r131+/BLiC/eR26+/τGFP mice.
Although the τGFP immunoreactivity is evident throughout the cell, the ISH signal, which reveals the location of Tas2r131 mRNA, is perinuclear, where rough ER is typically located. We in total inves-tigated 58 cells in 41 TB prepared from 3 Tas2r131+/BLiC/eR26+/τGFP mice. Occasionally, we observed a τGFP labeled cell that was not labeled by the riboprobe. One example is shown in Figure 3B (white arrow). Most likely, this is due to the low expression level of the Tas2r131 gene in heterozygous animals and/or the reduced sensitivity of the combined ISH/IHC protocol. Control experi-ments using wt mice verified the specificity of the anti-GFP antise-rum (Figure 3B,C). Taken together, our results demonstrate faithful expression of the recombinant Tas2r131 allele and show that the τGFP fluorescence reliably identifies Tas2r131-expressing cells in Tas2r131+/BLiC/eR26+/τGFP mice.
τGFP reporter gene expression is restricted to subsets of TRCs in TB of Tas2r131+/BLiC/eR26+/τGFP miceτGFP fluorescent cells were easily identified in TB of Tas2r131+/BLiC/eR26+/τGFP but not of wt mice (Figure 3B,C,D). To analyze whether the reporter gene expression is restricted to cells expressing taste
Figure 2. Expression analyses of the recombinant Tas2r131-BLiC allele. (A) RT-PCR: RNA, isolated from VP, FoP, FuP, SP, and NID was DNase I-digested and subjected to cDNA synthesis either including (+) or omitting (−) reverse transcriptase. Tas2r131-specific PCR products (508 bp) were detected in wt (Tas2r131+/+) and heterozygous (Tas2r131+/BLiC) mice. In contrast, PCR products specific for Cre (410 bp) were exclusively detected in heterozygous (Tas2r131+/BLiC) and homozygous (Tas2r131BLiC/BLiC) mice. As positive control (+) either genomic DNA (Tas2r131, β-actin) or plasmid DNA containing Cre coding sequence was used. (B) Colorimetric ISH: VP cross sections of wt (Tas2r131+/+) and homozygous (Tas2r131BLiC/BLiC) mice were hybridized with digoxigenin-labeled Tas2r131 and Cre riboprobes. Sections of Tas2r131BLiC/BLiC mice did not show any labeling when hybridized with Tas2r131 antisense (as) probe, indicating the absence of taste receptor RNA in KO mice. Hybridization of sections using a Cre-specific riboprobe exclusively labeled TB of homozygous gene-targeted mice.
receptors in the TB, we stained cross sections of VP of these mice by indirect IF using antisera against several taste cell marker pro-teins. Specifically, we analyzed the colocalization of τGFP protein with cells expressing the type I cell marker nucleoside triphosphate diphosphohydrolase 2 (NTPDase 2), the type II taste cell marker α-gustducin (α-Gust) and the type III cell marker aromatic amino acid decarboxylase (AADC). We detected τGFP fluorescence exclu-sively in a subset of α-Gust-expressing cells (Figure 3D) and did not find any τGFP-labeled (τGFP+) cells immunoreactive for NTPDase2 or AADC (Figure 3D). Our data demonstrate that the τGFP+ cells in the TB of Tas2r131+/BLiC/eR26+/τGFP mice represent a subset of type II TRCs.
Tas2r131 expression is initiated at different developmental stages depending on the type of TBTB development is initiated during embryogenesis and completed either neonatal (FuP) or postnatal (NID, FoP, VP), respectively (State and Bowden 1974; Harada et al. 2000; El-Sharaby et al. 2001; Amasaki et al. 2003; Zhang et al. 2006). To determine the onset of Tas2r131 expression in the developing TB, we systemati-cally investigated the postnatal expression pattern of the Tas2r131 receptor using the Tas2r131+/BLiC/eR26+/τGFP mice. We investigated its expression in lingual and palatal TBs at three different time points after birth (i.e., at P3, P9, P17). At P3, the newborn mice are not yet mobile, they are nursed, they do smell, however, their
Figure 3. Cre-mediated recombination in Tas2r131BLiC mice. (A) Breeding strategy to activate τGFP expression in Tas2r131 expressing cells. Coexpression of Cre recombinase with Tas2r131 leads to excision of the floxed stop cassette and the activation of ROSA26-driven τGFP expression in Tas2r131+/BLiC/eR26+/τGFP double knock-in mice. (B) Fluorescent ISH: VP sections of Tas2r131+/BLiC/eR26+/τGFP and control (Tas2r131+/+/eR26+/τGFP) mice were hybridized with a Tas2r131 specific riboprobe and subsequently immunohistochemically processed using an anti-GFP antiserum. In Tas2r131+/BLiC/eR26+/τGFP mice (n = 3), τGFP fluorescence was observed in Tas2r131-expressing cells. In a few cases, cells were labeled by the antiserum but not by the riboprobe (white arrow). τGFP was exclusively detected in gene-targeted, but not in wt mice. (C) IF: VP sections of Tas2r131+/BLiC/eR26+/τGFP and control (Tas2r131+/+/eR26+/τGFP) mice were processed using an anti-GFP antiserum. In Tas2r131+/BLiC/eR26+/τGFP mice, intrinsic τGFP fluorescence and the fluorescent labeling of the antiserum labeled the same cells. Fluorescent proteins were exclusively detected in double knock-in (Tas2r131+/BLiC/eR26+/τGFP) but not in control mice (Tas2r131+/+/eR26+/τGFP). (D) IF analysis of sections through the VP prepared from Tas2r131+/BLiG/eR26+/τGFP mice using antibodies against NTPDase2, α-Gust, and AADC. Fluorescent proteins were exclusively expressed in a subpopulation of α-Gust cells. VP of control mice (Tas2r131+/+/eR26+/τGFP) did not display any τGFP fluorescence.
Figure 4. Bitter taste receptor Tas2r131 and α-Gust are expressed at different postnatal developmental stages in gustatory tissues of mice. (A) Sections containing FuP, NID, SP, FoP, VP, and epiglottis of 3-, 9-, and 17-day-old (P3, P9, P17) Tas2r131+/BLiC/Rosa26+/τGFP and control mice were immunohistochemically processed using α-Gust antiserum. Colocalization studies of cells labeled by the intrinsic fluorescence of τGFP (green) with α-Gust (red), a marker for taste-like cells, were performed. Merged images visualize cells carrying exclusively τGFP protein (green), α-Gust (red), or both proteins (yellow). τGFP is detected at different time points of the mouse development. Although at P3 animals show τGFP fluorescence in TB of SP and FoP, expression of τGFP in FuP, NID, and VP was observed at P9 and in the epiglottis at P17. In these tissues, τGFP cells constitute a subpopulation of α-Gust cells. Asterisks denote unspecific autofluorescence. Scale bars 20 µm. (B) Schematic illustration summarizes the onset of Tas2r131 and α-Gust expression during development.
eyes and ears are still closed. At P9 the pups become more agile and are still nursed. P17 pups are shortly before weaning and get successively independent from their mothers (Silver 1995). At all 3 time points selected gustatory tissues (FuP, NID, SP, FoP, and VP) were prepared, sectioned, and immunohistochemically processed using the type II taste cell marker α-Gust (Figure 4A). Finally, IF for α-Gust and autofluorescence of τGFP were imaged simultane-ously in a laser scanning microscope. The onset of Tas2r131 and of α-Gust expression differed in TB depending on their location in the oral cavity. Whereas we did neither observe α-Gust nor Tas2r131 expression in TB of FuP, NID, or VP 3 days after birth, both genes are already expressed in TB of FoP and SP at this stage (Figure 4A). In contrast, at 9 and 17 days after birth TB in all locations show robust expression of both Tas2r131 and α-Gust. Furthermore, the number of Tas2r131- and α-Gust-expressing cells steadily increased with the age of the mice. Because not all α-Gust positive cells at all stages investigated were also positive for Tas2r131 we con-clude that Tas2r13-expressing cells constitute a subpopulation of α-Gust positive cells. Moreover, we investigated the hrGFP fluores-cence in the Tas2r131BLiG strain (Voigt et al. 2012) at P3 and P17 (Figure 5). A comparison of τGFP- and hrGFP-expressing cells in Tas2r131+/BLiC/eR26+/tGFP and Tas2r131BLiG/BLiG animals revealed similar Tas2r131 expression patterns in TB of FuP, NID, and VP. However, in tissue containing SP and FoP we observed τGFP+ cells (Tas2r131+/BLiC/eR26+/tGFP) but no hrGFP+ cells (Tas2r131BLiG/BLiG) 3 days after birth. Similar observations were obtained for epiglot-tis. At P17 fluorescent cells were observed in Tas2r131+/BLiC/eR26+/
tGFP but not in Tas2r131BLiG/BLiG mice. We assume that these varia-tions reflect the different expression levels of reporter proteins at this developmental stage because τGFP expression is controlled by the enhanced ROSA26 locus.
Identification of Tas2r131-expressing cells outside of the taste systemThe Tas2r131 receptor is expressed outside of the taste tissue (Voigt et al. 2012). Therefore, we analyzed cross sections of selected non-gustatory tissues prepared from Tas2r131+/BLiC/eR26+/τGFP mice. Strikingly, tissue sections of lung, trachea, ovary, geniculate ganglia (GG), trigeminal ganglia (TG), and the brain stem obtained from
Tas2r131+/BLiC/eR26+/τGFP mice showed single τGFP fluorescent cells (Figure 6). In contrast, we didn’t observe fluorescent reporter in tis-sue sections of these tissues using Tas2r131BLiG/BLiG mice (Figure 6) which carry a hrGFP fluorescent protein cassette directly under con-trol of the Tas2r131 promoter (Voigt et al. 2012). These data sug-gest that the binary genetic approach in the Tas2r131+/BLiC/eR26+/τGFP mice is more efficient visualizing Tas2r131-expressing cells com-pared with the direct knock-in strategy used in Tas2r131BLiG/BLiG mice. Alternatively, the fluorescent cells identified in these tissues in the Tas2r131+/BLiC/eR26+/τGFP mice might not acutely express Tas2r131 but rather reflect the history of activity of the Tas2r131 promoter.
Postnatal onset of Tas2r131 expression in nongustatory tissuesTo determine the onset of T2R expression in nongustatory tissues, we systematically investigated the postnatal expression pattern of the Tas2r131 receptor in the respiratory epithelium (RE), olfac-tory epithelium (OE), vomeronasal organ (VNO), thymus, trachea, lung, ovary, testis, and brain (Figures 6 and 7). At P3, we detected Tas2r131 expression in some of these tissues (NE, OE, VNO, thy-mus, trachea, testes, and brain) but not in others (lung, ovary). In lung and ovary, Tas2r131 expression was first observed at P9. Similarly, α-Gust expression was observed in most (NE, OE, thymus, trachea, and lung) but not all (not in VNO, ovary, testis, and brain) investigated tissues at P3. The VNO contained α-Gust cells at P9, in contrast we did not observe α-Gust in testis and brain even in P17 animals.
We detected Tas2r131 expression in lung at P9 and P17, whereas the α-Gust protein was already detected at P3. Vice versa in VNO we found Tas2r131 expression already at P3 whereas the presence of α-Gust protein was first noticed at P9. The number of Tas2r131- and α-Gust-positive cells steadily increased during postnatal devel-opment. However, colocalization studies of Tas2r131 and α-Gust expressing cells in nongustatory tissue revealed that the Tas2r131 cells do not always express this G protein (Figure 7: NE [P17]; OE [P3, P9, P17]; VNO [P3, P17]; Figure 8: trachea [P3]; lung [P9, P17], and thymus [P9, P17]). Remarkably, the majority of the investi-gated nongustatory tissues showed Tas2r131 expression before P3,
Figure 5. hrGFP fluorescence in gustatory tissues of Tas2r131BLiG mice at P3 and P17. Tissue sections containing FuP, NID, SP, FoP, and VP of 3- and 17-day-old (P3, P17) Tas2r131BLiG/BLiG mice show intrinsic hrGFP fluorescence in P17, but not in P3 animals. hrGFP fluorescence in tissue containing epiglottis was not observed at either developmental stage. Scale bar 20µm.
preceding its expression in most gustatory tissues analyzed (FuP, NID, and VP) (Figure 4). Other nongustatory tissues analyzed in the Tas2r131+/BLiC/eR26+/τGFP mice at P17 (including eye, lymph nodes,
thyroid gland, esophagus, liver, kidney, spleen, pancreas, brown and white adipose tissue, skeletal muscle, heart, and blood) did not con-tain τGFP fluorescent cells (not shown).
Figure 6. Cre-mediated recombination in nongustatory tissues in Tas2r131 gene-targeted mice. Tas2r131 expression, visualized by reporter fluorescence (green), was analyzed in 14 µm cryosections of lung, trachea, ovary, GG, TG, as well as in the brain of gene-targeted mice. To visualize the tissue structure, background fluorescence of the tissue is enclosed in the pictures (white). Single τGFP cells (white arrowheads) were observed in Tas2r131+/BLiC/eR26+/τGFP mice. Scale bar 20 µm.
Figure 7. Expression analysis of bitter taste receptor Tas2r131 and α-Gust in the nose at different postnatal stages in mice. (A) Tissue sections containing the RE, OE, or VNO of 3-, 9-, and 17-day-old (P3, P9, P17) Tas2r131+/BLiC/Rosa26+/τGFP and control mice were treated with α-Gust antiserum. Merged images visualize cells positive for τGFP (green), α-Gust (red), or both proteins (yellow). In gustatory tissue, τGFP protein is detected at different time points of the mouse development. Tissues show initial τGFP protein at different time points (P3: NE, OE, VNO). A subpopulation of τGFP cells expresses α-Gust. Note additional τGFP-positive cells which do not express α-Gust. Scale bars 20 µm. (B) Onset of Tas2r131 and α-Gust expression in the nose.
The Tas2r131BLiC strain is a valuable tool to manipulate Tas2r131-expressing cells. Using a binary genetic strategy to characterize Cre-mediated recombination in these animals, we show that τGFP
reliably visualizes Tas2r131 cells by intrinsic reporter fluorescence. As expected, τGFP is faithfully expressed in a subset of TRCs. The intrinsic fluorescence is exclusively observed in α-Gust cells, con-firming earlier studies colocalizing Tas2rs with α-Gust (Kim et al. 2003; Behrens et al. 2007; Voigt et al. 2012). We observed Tas2r131
Figure 8. Tas2r131 and α-Gust expression at different postnatal stages in trachea, lung, thymus, ovary, testis, and brain. (A) Sections obtained from trachea, lung, thymus, ovaries, testis, and brain of 3-, 9- and 17-day-old (P3, P9, P17) Tas2r131+/BLiC/Rosa26+/τGFP and control mice processed with α-Gust antiserum. Colocalization identifies cells labeled by τGFP (green), α-Gust (red), or both (yellow). Several nongustatory tissues start to express τGFP at different time points (P3: trachea, thymus, testis, and brain; P9: lung, ovaries). Scale bars 20 µm. (B) Summary of the onset of Tas2r131 and α-Gust expression in trachea, lung, thymus, ovary, testis, and brain at several developmental stages.
expression in FuP, NID, SP, FoP, and VP in the oral cavity, which is in agreement with previous work describing Tas2r131 cells mainly in FoP and VP, but also in TB of the anterior region of the tongue (FuP) and in the palate (NID, SP) (Voigt et al. 2012). Although no reporter fluorescence was observed in, for example, lung, trachea, ovaries, ganglia and brain of Tas2r131BLiG/BLiG mice, individual τGFP cells were present in these tissues in Tas2r131+/BLiC/eRosa26+/τGFP mice. Of note, the expression level of the reporter protein is determined by the ROSA26 locus in these animals.
Although Tas2rs in TRCs detect bitter-tasting compounds and therefore protect against the ingestion of harmful substances, Tas2rs present in other cell types in the body may recognize noxious com-pounds and initiate defense mechanisms for their effective elimina-tion (Green 2012). Because Tas2rs seem to play an important role in the survival of an individual, we analyzed the onset of Tas2r131 expression in both gustatory and nongustatory tissues. We found that Tas2r131 cells in TB of the oral cavity are expressed at early time points, however not uniformly across all types of TBs. TRCs in FoP and SP are the first which are equipped with Tas2r131 and the signaling molecule α-Gust, followed by TRCs in FuP, NID, and VP. Interestingly, whenever TRCs express this bitter taste receptor, the taste signaling molecule α-Gust is always coexpressed. The simulta-neous expression of Tas2rRs and α-Gust in TRCs of newborn mice is similar to the expression pattern reported for adult mice (Adler et al. 2000; Kim et al. 2003). Moreover, Tas2r131 cells in newborns already constitute a subpopulation of α-Gust-expressing cells, again reflecting the scenario observed in adult animals (Adler et al. 2000). We suspect that the additional α-Gust cells are sweet or umami responsive cells, because those cells are also immunoreactive for α-Gust (Wong et al. 1996; He et al. 2004), or additional bitter responsive cells, because Tas2r131 cells comprises about half of the entire population of bitter sensor cells (Voigt et al. 2012). The putative presence of sweet and umami responsive cells in the newborn mice would not be surprising as mammals are congenitally fed with milk known to contain the sweet tasting compound lactose as well as free glutamate.
Surprising to us however was that we did not detect Tas2r131 receptor expression at P3 in VP, the site in the oral cavity known to carry the majority of Tas2r131 cells in adult mice (Voigt et al. 2012), whereas TB of FoP and SP already contained Tas2r131 cells at that stage. TB development in the VP is still in progress at P3. Even though nascent vallate trenches are conspicuous at birth, nearly all of the TBs mature postnatally in mice (Cooper and Oakley 1998). Although the number of TB increases shortly after birth (P2–P6), their maturation is still in progress (State and Bowden 1974). At P8, vallate TBs appear for the first time with their typical spindle-shaped morphology and a gustatory pore and contain TRCs immunoreac-tive for α-Gust (State and Bowden 1974; Bigiani et al. 2002). We observed Tas2r131 and α-Gust cells at P9, corroborating the α-Gust data reported by Bigiani et al. (2002). We also observed an increase in the number of Tas2r131 and α-Gust cells between P3 and P17 in all types of TBs, which again is in accordance with previous work showing an increase in the number of α-Gust cells from P4 to P14 (Bigiani et al. 2002). Directly after birth, milk is the only food source for rodents and bitter detection might thus not have the highest pri-ority at this stage. However, when milk is replaced by solid food starting at about 2 weeks of age in rodents, the ability to detect bitter stimuli may be indispensable for survival. Indeed, our data demon-strate that at least one member of the Tas2r-family and the taste associated G protein α-Gust is coexpressed in TRCs by then.
Identity and function of extra-oral cells expressing Tas2rs are not well understood and so far no data reporting expression
of these receptors during murine development are available. We found Tas2r131 expression in extra-oral sites shortly after birth. At P3, Tas2r131 was expressed in the respiratory (RE, OE, VNO, and trachea), lymphatic (thymus), reproductive (testis), and the peripheral and central nervous system (brain). Within the second week after birth additional tissues belonging to the respiratory (lung) and reproductive (ovaries) system were identified to con-tain Tas2r131 cells. We observed that the number of Tas2r131 cells in those extra-oral sites increases within the first weeks after birth. In the nose, we detected spindle-shaped as well as bipolar Tas2r131-τGFP cells in both the VNO and the OE, indicating the expression of Tas2r131 in 2 morphologically distinct cell types. However, in tissues of the lower respiratory tract such as trachea and lung, we solely observed scattered spindle-shaped Tas2r131-τGFP cells, indicating that expression is restricted to one cell type. Interestingly, we also saw α-Gust immunoreactivity exclu-sively in cells with spindle-shaped morphology and not in bipolar Tas2r131-τGFP cells. Based on morphology and immunoreactivity we suspect the bipolar cells to be chemoreceptor neurons. These extend a single dendrite that reaches up to the surface of the tissue and ends in a knob-like swelling from which cilia arise (Firestein 2001). Chemoreceptor neurons send axons directly to the fore-brain and trigger behavioral responses to general odors includ-ing food and predator odors (Firestein 2001). The spindle-shaped Tas2r131-τGFP cells are α-Gust immunoreactive. Because α-Gust is a marker of chemoreceptive cells and these cells have a typical pear- or flask-shape morphology we speculate that these Tas2r131-τGFP cells are solitary chemosensory cells (SCCs), which have been reported to be scattered along the respiratory tract from the nasal cavity to bronchi (Finger et al. 2003; Sbarbati et al. 2004; Merigo et al. 2005; Kaske et al. 2007; Lin et al. 2008). SCCs have analogies with TRCs as they express Tas2rs and correspond-ing signaling proteins (α-Gust, PLCβ2, TRPM5, and IP3R3) and respond to various chemical stimuli including bitter compounds (Gulbransen et al. 2008; Ogura et al. 2010). However, SCCs are not aggregated in buds (Finger et al. 2003; Sbarbati et al. 2004; Merigo et al. 2005). SCCs are distributed with high density in strategic areas of the body (i.e., respiratory- and GI tract) and are part of the so-called diffuse chemosensory system, which is sup-posed to be involved in the recognition and processing of diverse environmental cues (Sbarbati et al. 2010). The Tas2r131BLiC strain will help to dissect the functional role of these and other Tas2r131 cells in both gustatory and nongustatory tissues in the future.
Funding
This work was supported in part by the Deutsche Forschungsgemeinschaft (BO 1743/2 to U.B.).
AcknowledgmentsWe thank Olaf Pongs for continuous support. We thank Stefanie Demgensky, Johanna Walther, and Josefine Würfel for expert technical assistance and Ali Derin and Jasmin Mattern for excellent animal care.
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